Immunofluorescence Studies
Six rabbits were used in this sub-study. The animals were anesthetized with an overdose of pentobarbital sodium (30 mg/kg intravenously) and anticoagulated with heparin (180 IU/kg). After median sternotomy, the hearts were quickly excised. Then tissues were obtained from the SVC, PVs, RA and LA, fixed in 4% paraformaldehyde until embedment in paraffin. Four-micrometer sections were cut from paraffin blocks of these tissues. The samples were incubated with primary antibodies against c-TNT (1:100, Abcam, UK), KCa3.1 antibody (Bioss, 1:200) overnight and then sections were subsequently with secondary antibody, fluorescein isothiocyanate conjugated Affinipure Goat Anti-rabbit IgG (Abcam, 1:50) and Fluorescein (CY3)-conjugated Affinipure Goat Anti-mouse lgG (Aspen, 1:50). Cell nuclei was stained with 4′,6-diamidino-2-phenylindole. Image Pro Plus 6.0 (Media Cybernetics, Inc., Rockville, Maryland) was used to give quantitative analysis. KCa3.1 expression in myocytes were calculated by the sum of KCa3.1 density value in CTNT marked area. Three visual fields at 400×magnification were then chosen randomly from each sample to assessment.